Table 2. Specific activity and hydrolysis/transglycosylation ratio of wild-type GTFO-∆N and GTFA-∆N enzymes, and of GTFO-derived mutants, at 100 mM sucrose, and methylation, ¹H NMR and pullulanase digestion analysis of the polysaccharides produced.

Enzyme	Activitya (U/mg)	Hydrolysis/trans-glycosylation (%)	Chemical shiftc(%)		Methylation analysisb(%)				Maltosed
			(α1 → 6)	(α1 → 4)	Glcp(1 →	→ 4)Glcp(1 →	→6)Glcp(1→	→4,6)Glcp(1→
GTFA-ΔN	22.7 ± 1.8	9/91	42	58	13	45	27	15	45.9 ± 2.2
GTFO-ΔN	13.7 ± 0.9	43/57	21	79	11	69	9	11	8.7 ± 1.3
GTFO-ΔN A977Q	14.4 ± 1.2	35/65	31	69	16	51	18	15	24.9 ± 3.2
GTFO-ΔN lp977	1.5 ± 0.3	35/65	33	67	14	53	18	15	25.5 ± 2.0
GTFO-ΔN SS-HA	11.0 ± 1.5	44/56	24	76	12	68	10	10	8.3 ± 1.0
GTFO-ΔN α4	13.3 ± 2.6	37/63	36	64	19	43	21	18	28.1 ± 2.4
GTFO-ΔN A977Q/α4	12.2 ± 1.2	33/67	39	61	18	42	22	18	35.8 ± 3.0
GTFO-ΔN lp977/α4	1.2 ± 0.3	30/70	40	60	17	44	22	17	31.6 ± 3.1
GTFO-ΔN SS-HA/α4	13.4 ± 0.9	37/63	36	64	16	45	22	17	29.1 ± 3.5
GTFO-ΔN lp977/SS-HA	0.3 ± 0.08	36/64	31	69	13	56	19	12	27.0 ± 3.3
GTFO-ΔN lp977/SS-HA/α4	1.9 ± 0.4	30/70	40	60	17	44	23	16	34.1 ± 2.8
GTFO-ΔN A977Q/SS-HA/α4	15.8 ± 2.2	31/69	39	61	15	45	23	17	35.4 ± 3.6
GTFO-ΔN A977Q/V1083D	17.7 ± 1.5	34/66	36	64	18	42	22	18	28.8 ± 2.5
GTFO-ΔN A977Q/G1087N	29.0 ± 3.0	31/69	34	66	16	47	20	17	25.2 ± 2.1
GTFO-ΔN A977Q/V1083D/G1087N	21.9 ± 1.2	30/70	38	62	17	42	22	19	30.3 ± 2.8

^aActivity measured at 100 mM sucrose at 35 °C. ^bThe ratios of the different glucosyl substitutions are shown in molar percentage based on the intensities from GLC. ^cThe ratios of integration of the surface areas of the (α1 → 6) linkage signal at 4.96 ppm and the (α1 → 4) linkage signal at 5.36 ppm in the ¹H NMR spectra are displayed. ^dThe percentage of maltose in the pullulanase M1 digestion product mixtures of polysaccharides produced by the respective enzymes based on the glucosyl unit.